Tropomyosin is a protein existing in all eukaryotic cells, with multiple isoforms encoded by at least 4 separate genes. The role of this protein is best understood in striated muscle, where, in association with the tropinin complex, TM renders calcium sensitivity to the interaction between actin and myosin. Its role is much less well understood in smooth muscle, where TM is found on microfilaments but the degree of phosphorylation of myosin light chain appears to regulate the interaction between actin and myosin. Differences in degree of actin binding between different non-muscle TM isoforms suggests that different isoforms of TM may associate with act in to form discrete and functionally different populations of microfilaments. Examining the assembly of TM isoforms into microfilaments from different cell types important to the cardiovascular system offers the possibility of understanding how the developing contractile apparatus is put together. Perhaps a better understanding of mechanisms of normal assembly during development will shed new insight on processes that can be disturbed, resulting in abnormal development. This project proposes to investigate the mechanism of assembly of TM into micro-filaments using pulse-chase experiments and pulse experiments with 3H-puromycin to identify nascent chains in cultured cells from muscle (cardiac, skeletal, and smooth), and fibroblasts. Differentiating isoform-specific versus cell-type specific differences in assembly mechanism will be addressed by transfecting wild-type TM isoforms into cells not normally expressing the transfected isoform, followed by examining the assembly of this isoform into microfilaments by immunofluorescent microscopy, purification of the thin filaments from labeled cells, and localization of nascent TM chains with the use of puromycin. Functional domain of the TM molecule that are critical for proper assembly into microfilaments will be investigated by transfecting deletion mutant TMs into cultured cells and examining microfilaments formed by immunofluorescence microscopy. Important information about the assembly of microfilaments during differentiation can be gathered during the course of this project. At least as important will be the protected education of the applicant for a prolonged period, increasing the likelihood of a successful subsequent research career.